DNA and RNA constitute the key molecular components of all genetic processes, and have similar structural components. DNA typically exists as a complex of two anti-parallel linear strands or sequences of deoxyribonucleotide structural units, each unit of which consists of a nitrogenous base (adenine (A), thymidine (T), cytosine (C) or guanine (G)), a pentose sugar (a 5-carbon sugar), and a phosphate group. RNA is typically single stranded, and uses uracil (U) in place of thymidine (T). Moreover, the pentose sugar in DNA is 2-deoxyribose, while the pentose sugar in RNA is ribose. The nitrogenous bases of DNA and RNA are of two classes: the larger nine-member double-ring purines, A an G, and smaller six-member single-ring pyrimidines, C, T and U.
According to the Watson-Crick paradigm, a double helix is formed from two strands of DNA, which are paired by hydrogen bonding between complementary nitrogenous base pairs. The rules of base pairing require size complementarity—large purines pair with small pyrimidines. A further basis of complementarity is the geometrical (or spatial) correspondence of hydrogen bond donors and acceptors present on the nitrogenous bases: The G-C pair has three hydrogen bonds that pair, and the A-T base pair has two hydrogen bonds that pair, in such a manner that hydrogen bond donors pair with hydrogen bond acceptors. Other possible base pairings are said to be “mismatched”. “Mismatches” between nucleotide bases result in greater instability relative to perfectly matched nucleotide bases. Instability caused by nucleotide base mismatches can be detected by means of comparing melting (disassociation) temperatures of hybridized DNA strands, and is the current basis of many forms of genetic testing.
Alternative base pairing systems utilizing only purine nucleobases have been investigated. For example, although a duplex from polyinosine and polyadenosine oligonucleotides through Watson-Crick interaction of inosine and adenosine (two purine nucleobases) has previously been reported (Rich, Nature 181, 521-525 (1958)), subsequent studies showed that the complex was actually a triplex with no independent Watson-Crick duplex present. (Howard et al., Biochemistry, 16, 4647-4650 (1977). Another study describes weak interaction between polyinosine and a substituted polyadenosine, and speculates that more than one mode of interaction was likely present, including possibly duplex formation through Watson-Crick interaction, although the data was inconclusive. (Howard et al., Biochemistry, 16, 4637-4646 (1977). Another study described duplexes formed through interaction of purine-purine dyads based on non-natural backbones with modified non-ribosyl sugars (homo-DNA, consisting of hexopyranosyl-(6′→4′)-oligonucleotides); the authors further stated that duplex formation is not possible in purine-purine duplexes having natural backbones (Groebke et al., Helvetic Chimica Acta, Vol. 81, 1998). Non-Watson-Crick pairing all-purine duplexes with repeating sequences have also been reported. (Howard et al., J. Biol. Chem. 250, 3951-3959 (1975)) (Howard et al., Biochemistry, 16, 4637-4646 (1977)).
More recently, formation of duplexes with a natural ribosyl backbone pairing purines with unnatural nucleobases and pyrimidines with additional unnatural nucleobases has been reported (Gao et al., Angew. Chem. Int. Ed. 44, 3118-3122 (2005).
Notwithstanding the failure in the prior art to generate oligonucleotides comprising purine-purine dyads, purine-purine duplexes could potentially be useful for hybridization in nucleic acid-based diagnostic assays. For example, nucleic acid sequences containing a plurality of purine-purine regions could be enzymatically amplified and targeted by purine-purine probes. Polypurine regions of nucleic acid sequence could be replicated by natural polymerases pairing pyrimidines with purines to yield complementary polypyrimidine sequences, and could also be specifically targeted for hybridization with completely different complementary all-purine sequences. In addition, purine-purine duplexes could be used in situations where canonical Watson-Crick duplexes may be affected by common processes of molecular biology. Duplexes of purine-purine nucleic acids will not act as substrates in many enzymatically-mediated processes, such as polymerase replication and amplification, ligation, restriction, and mismatch repair, which depend on recognition of a duplex nucleic acid. For example, oligonucleotides with capture sequences complementary to oligonucleotides immobilized on a surface could be used to form all-purine duplex structures, which would be inert in the presence of these enzymatically-mediated processes. Small molecules that target duplex nucleic acids could also exhibit altered interaction with purine-purine duplexes, thereby allowing, for example, dyes such as SYBR Green I to be used to specifically detect formation of a canonical duplex in solution with purine-purine duplexes performing a hybridization function that remains undetectable in monitoring fluorescence of the dye.
Accordingly, there is a need in the art for nucleotide duplexes comprising complementary purine-purine nucleobases.